Role of carbohydrate moiety of IL-5. Effect of tunicamycin on the glycosylation of IL-5 and the biologic activity of deglycosylated IL-5.
IL-5 is a T cell-derived lymphokine that induces B cell growth and differentiation in murine systems. In this study, we examined the role of carbohydrate moiety of IL-5 in the expression of biological function. IL-5 polypeptides translated in Xenopus oocytes were heterogeneous in terms of isoelectric point (pI 4.7 to 8.0) and m.w. (45,000 to 60,000 under nonreducing conditions) and yielded m.w. of 25,000 to 30,000 under reducing conditions. Treatment of rIL-5 with N-glycanase under reducing conditions yielded an IL-5 monomer of m.w. 12,000 to 14,000. Furthermore, deglycosylated rIL-5 that had been translated in the presence of tunicamycin showed very limited heterogeneity by two-dimensional gel electrophoresis (first dimension, nonequilibrium pH gradient electrophoresis; second dimension, SDS-PAGE). The m.w. was 27,000 to 28,000 under non-reducing conditions and migrated to m.w. 13,000 to 14,000 under reducing conditions. These results indicate that IL-5 is a glycoprotein carrying the N-glycosidically-linked carbohydrates. Treatment of IL-5 with sialidase caused the decrease in the heterogeneity in isoelectric point of IL-5. Deglycosylated rIL-5 that had been obtained from tunicamycin-treated oocytes could bind to IL-5-responding cells (T88-M), which express both high- and low-affinity IL-5 receptors, as efficient as intact rIL-5 under high-affinity conditions. Scatchard plot analysis of equilibrium binding of 35S-labeled rIL-5 to T88-M cells revealed that the dissociation constants (Kd) of glycosylated rIL-5 and deglycosylated rIL-5 were 127 pM and 110 pM, respectively. IL-5 activities determined by both B cell growth and differentiation assays were not affected by deglycosylation. These results indicate that N-linked glycoside moiety of IL-5 molecules may not play an essential role in the expression of its activity.
Tominaga
A
A
Department of Biology, Kumamoto University Medical School, Japan.
Takahashi
T
T
Kikuchi
Y
Y
Mita
S
S
Naomi
S
S
Harada
N
N
Yamaguchi
N
N
Takatsu
K
K
D000818:Animals; D001681:Biological Assay; D015180:Electrophoresis, Gel, Two-Dimensional; D006026:Glycoside Hydrolases / Q000494:pharmacology; D006031:Glycosylation; D007075:Immunoglobulin M / Q000096:biosynthesis; D066298:In Vitro Techniques; D015848:Interleukin-5 / Q000378:metabolism / Q000494:pharmacology*; D008213:Lymphocyte Activation / Q000187:drug effects; D008970:Molecular Weight; D009439:Neuraminidase / Q000494:pharmacology; D011971:Receptors, Immunologic / Q000378:metabolism; D018123:Receptors, Interleukin; D053649:Receptors, Interleukin-5; D011994:Recombinant Proteins; D013329:Structure-Activity Relationship; D014415:Tunicamycin / Q000494:pharmacology
D007075:Immunoglobulin M; D015848:Interleukin-5; D011971:Receptors, Immunologic; D018123:Receptors, Interleukin; D053649:Receptors, Interleukin-5; D011994:Recombinant Proteins; D014415:Tunicamycin; D006026:Glycoside Hydrolases; C020668:glycanase; D009439:Neuraminidase